The overriding objective of this R21 application is to develop a new innovative paradigm and address a new approach to nonhuman primate models of human congenital diseases using breakthrough technologies in genetic engineering. Mice have evolved as a premiere model for human genetics largely because of the ability to manipulate their genome with gene targeting, which has enabled substantial contributions to the understanding of genetics and disease. With the advent of more advanced methods for gene editing, including clustered regularly interspaced short palindromic repeats (CRISPR/Cas9), it is now possible to modify the genetics of other species that are more similar to humans. However, CRISPR/Cas9 gene editing in the nonhuman primate germline is very inefficient and laborious, and it is unclear that such methods will find widespread use for providing improved models of human disease. The objective of these studies is to capitalize on our innovative capabilities for in vivo delivery of gene editing tools in rhesus monkeys. The Specific Aims for these studies will: (1) Benchmark knock-out frequencies, and (2) Benchmark knock-in frequencies in the developing brains of early gestation fetal rhesus monkeys. The outcome of these studies will demonstrate the feasibility of this approach for the future production of translational models of human congenital diseases that will be predictive for modeling patient-derived mutations and assessing new therapeutics for human use.